S. pneumoniae (or pneumococcus) and the discovery of the "Transforming Principle"
By the middle of the twentieth century, Gregor Mendel's principals of genetic inheritance had been widely accepted within the scientific community, but the chemical composition of Mendel's so called "hereditary units" remained an enigma to scientists. Scientists eventually recognized that genes and their respective alleles (the units of inheritance) could be found on the chromatin within the human nucleus, but then the question became whether DNA or its associated proteins was the basis of inheritance. Most researchers were inclined to believe that protein was the hereditary material as previous chemical analyses had shown that proteins were more variable chemically, physically and functionally than DNA. It thus came as a great shock to the scientific community when DNA was identified as the basis of inheritance (4). Three experiments were absolutely essential in the generation and acceptance of this new biological paradigm: Griffith's experiment in 1928, Avery et al. experiment in 1944, and Hersey and Chases' experiment in 1952. Two of these three experiments utilized the microbial pathogen Streptococcus pneumoniae and will be discussed in detail here. Griffith's Experiment By the late 1920's, the quest for the genetic material responsible for inheritance had reached the molecular level. The question playing on the minds of the scientific community was what genetic component of chromosomes, DNA or its associated proteins, is responsible for inheritance (5)? Frederick Griffith, a British microbiologist, discovered the phenomenon of bacterial transformation purely by accident in 1928, though he was unfortunately unable to discover the chemical composition of this "transforming principal" beyond the fact that it was able to somehow survive treatment with intense heat (4). In the aftermath of the 1918 flu pandemic, Griffith (like many other microbiologists at the time) was attempting to develop viable vaccines that could help mitigate the burden of infectious diseases in Europe and internationally; Griffith chose to focus his attention on a Streptococcus pneumoniae vaccine as S. pneumoniae had been a major cause of bacterial pneumonia in the late 19th century (4). Griffith was working with two strains of S. pneumoniae that were distinct morphologically and in terms of virulence. One of the strains Griffith used in his experiments possesses a smooth, polysaccharide capsule (S strain) while the other strain utilized by Griffith lacks a polysaccharide capsule, causing it to appear rough morphologically (R strain). The R strain is nonvirulent and incapable of causing disease in a health host, but the S strain displays considerable virulence and is a significant human pathogen (4 5) . Griffith noted that mice injected with S strain microbes died several days later of pneumonia, while mice injected with R strain microbes experienced no disease as a result of the injection. Griffith hypothesized that he could render the S strain nonvirulent through heat treatment. He later later affirmed this hypothesis through experimentation: mice injected with the heat-treated S strain did not develop pneumonia, just as he had expected (3). When he injected mice with heat-treated S strain and wild type R strain Streptococcus pneumoniae however, the mice expired several days later after developing pneumonia. Baffled, Griffith dissected his mice in an attempt to isolate the etiological agent responsible. When Griffith stained the culpable microbes from the blood of the deceased mice, he was amazed to find himself looking at S. pneumoniae bacteria with a polysaccharide coat (S strain). Based on these observations, Griffith postulated that a chemical component from the virulent S strain had somehow survived the heat treatment and had gone on to transform the innocuous R strain into the more pernicious S strain. Griffith later cultured the S. pneumoniae that he had previously isolated and injected them into another cohort of mice. These mice also contracted pneumonia and died within several days of the injection. Once again, Griffith isolated the etiological agent responsible for the demise of the mice and once again, Griffith encountered the polysaccharide capsule characteristic of the S strain. This lead Griffith to conclude that whatever transformation had occurred was inheritable and stable (1). With our current understanding of genetics and microbiology, we can infer that the DNA of the heat-treated S strain was able to renature within the mouse and that this now naked DNA was taken up by competent R strain bacterium in a process we still describe as transformation (4). Unfortunately, this knowledge was unavailable to Griffith and he was never quite able to describe the mechanism of the bacterial transformation he had observed. Avery, MacLeod, and McCarty's Experiment Oswald Avery and his associates at Rockefeller University in New York had begun working with S. pneumoniae several years before the publication of Griffith's work with the "transforming principle" in 1928. Prior to the dissemination of Griffith's research, Avery and his colleagues were preforming comprehensive biochemical and structural analyses of the pneumococcal cell capsule and its role in microbial virulence. Avery and his team were able to correctly infer that an intact polysaccharide capsule is an essential virulence factor for S. pneumoniae (as this capsule protects the microbe from the host's various immunological defense mechanisms) and that the chemical composition of a bacterium's polysaccharide capsule could be used to classify the bacterium (2, 4). When Griffith's work was first brought to the attention of the Rockefeller research team, they quickly recognized its importance and decided to apply their considerable expertise in a quest to uncover the precises chemical composition of the "transforming principle" that was capable of reverting a non-encapsulated bacterium back to its original encapsulated form. To discern the precise biochemical composition of the "transforming principle", the Rockefeller research team employed the tried and true process of elimination.Avery and research associates MacLeod and McCarty recognized that the transforming agent they sought could be either DNA, RNA or protein. Avery's research team also acknowledged the possibility that the polysaccharide capsule could simply reassemble around the non-encapsulated R strain. Such an event would represent a morphological change but not a genomic modification. Unlike Griffith, Avery, MacLeod and McCarty transformed their S. pneumoniae in culture rather than in living mice, a technique that allowed the researchers a greater degree of control over their experiment (1,4). Procedure Avery and his colleagues first killed clonal S. pneumoniae cells using heat. These cells were treated with detergent, which damaged the integrity of the phospholipid bilayer, resulting in cell lysis. The solution was centrifuged and the lysate (which contained the DNA, RNA, protein and carbohydrates of interest) was decanted off into a several different test tubes to be used in future transformation assays. Avery, MacLeod and McCarty first demonstrated that the lysate could indeed induce transformation by applying the lysate to a test tube containing the non-encapsulated R strain, which subsequently developed the polysaccharide capsule characteristic of S strain Streptococcus pneumoniae. After it had been established that the transforming principal could be found in the lysate collected, the lysate was incubated with the enzyme SIII, which catabolized the polysaccharides present. This now polysaccharide-free lysate was then added to another R strain test tube culture, which subsequently transformed into the more virulent S strain. This proved definitively that the polysaccharide capsule was not just simply reforming around the R strain microbes present and that there was indeed some sort genomic metamorphosis occurring, as Griffith had indicated when he proved that the transformation he had induced was inheritable. The lysate remaining after the SIII treatment was then treated with 2 specific types of proteases (trysin and chymotrypsin), which digested all residual proteins within the lysate. This now polysaccharide and protein free lysate was added to yet another R strain test tube and once again, the researchers witnessed a transformation event (1). This indicated that protein was not the transforming principle Avery and his team were seeking. The nucleic acids present in the remaining lysate was precipitated out of solution using pure-grain alcohol and purified through a series of washes with chloroform. Once the nucleic acids were purified they were re-suspended and partitioned into 2 separate test tubes. One test tube was subjected to DNase enzymatic treatment while the other was treated with RNase so that RNA and DNA could be isolated, respectively. When added to R strain Streptococcus pneumoniae cultures, only the lysate nucleic acids that had been treated with RNase had any metamorphic ability. As RNase degrades RNA and presumably only DNA remained in this solution, Avery and his fellows theorized that DNA was the transforming principle they sought. As a final test, the Rockefeller research team then added DNase to the nucleic acid solution that had previously induced transformation and added this resulting mixture to a R strain culture. The R strain remained unchanged, thus proving the hypothesis generated by Avery and his colleagues correct. The additional confirmatory tests preformed to confirm the biochemical composition of the transforming principal (e.g. qualitative tests, chemical analyses comparing the ratio of nitrogen to phosphorus, and immunological assays) all confirmed the presence of DNA (1, 4). Today, we view Avery's results as indisputable evidence that DNA is the molecule of inheritance and the causative agent behind transformation, but unfortunately Avery's results were not well received by the scientific community upon their publication in 1944. This is most likely because most scientists were still convinced that proteins, not DNA, held the key to unlock the genetic code, despite all evidence to the contrary. The task of convincing the scientific elite that DNA was of pivotal importance fell upon future researchers like Hersey and Chase. Though initially poorly received, the manuscript published by Avery, MacLeod and McCarty in 1944 ("Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types”) is now considered to be a masterful account of an expertly designed experiment and definitive proof that DNA is the "transforming principle" (2, 4). References 1. DNA Learning Center 2. Oswald Avery’s Pneumococcus Experiments: Forerunner of the DNA Story 3. Famous DNA Experiments 4. Isolating Hereditary Material: Frederick Griffith, Oswald Avery, Alfred Hershey, and Martha Chase 5. Preforming Griffith's Experiment